Neutral sphingomyelinase

ABSTRACT

The present invention relates to eukaryotic neutral sphingomyelinase (nSMase) and its application.

This is a 371 of PCT/EP 98/05127 filed Aug. 11, 1998, which claimsbenefit under 35 USC 119(e) to U.S. provisional application No.60/078,386, filed Mar. 18, 1998.

The present invention relates to nucleic acids coding for eukaryoticneutral sphingomyelinase, and applications thereof.

Sphingomyelin is an essential component of plasma membranes. Degradationof sphingomyelin gives a number of substances having potential secondmessenger properties, e.g., ceramide, sphingosine,sphingosine-1-phosphate. Two sphingomyelin-cleaving enzymatic activitiesare known, namely that of lysosomal acid sphingomyelinase, and that ofplasma-bound neutral sphingomyelinase.

Bacterial neutral sphingomyelinase is a secreted soluble protein.

The present invention for the first time provides nucleic acids/codingfor eukaryotic neutral sphingomyelinase. Eukaryotic neutralsphingomyelinase (nSMase) is characterized in that it cleavessphingomyelin into ceramide and phosphocholine and that its activitydepends on the addition of magnesium ions. It is a membrane-boundenzyme. Its maximum activity is achieved in the neutral pH range.

FIG. 1 shows the gene sequence of human neutral sphingomyelinase.

FIG. 2 shows the gene sequence of murine neutral sphingomyelinase.

FIG. 3 shows the results of the Northern and Western blotting ofnSMase-overexpressing cell lines.

FIG. 4 shows the strategy for producing murine knockout mutants. Theletters designate restriction sites.

FIG. 5 shows constructs for obtaining transgenic mouse mutants.

Preferably, the nucleic acid according to the invention is a nucleicacid coding for the neutral sphingomyelinase of a mammal. Morepreferably, it codes for human or murine neutral sphingomyelinase. Thecorresponding nucleic acid sequences are disclosed as SEQ. ID. NO. 3 andSEQ ID. NO. 4, respectively.

Parts of the nucleic acid sequences are identical with the EST sequencesAA028477 and AA013912 (murine) and W32352 and AA056024 (human).

When he knows the amino acid and nucleic acid structure of human andmurine neutral sphingomyelinase, one skilled in the art can easilydetect the corresponding nucleic acids and proteins from othereukaryotes, considering the high homology between human and murinenSMases. To do this, he can either use cross-reacting antibodies for apurification by specific affinity chromatography, or he can synthesizeoligonucleotide primers on the basis of the nucleic acid sequence andamplify the desired nucleic acids in a cDNA library of the eukaryoteusing polymerase chain reaction. The corresponding cDNA library can beobtained in a per se known manner by isolating mRNA from a tissuesample, followed by reverse transcription. From the nucleic acidsequence, the amino acid sequence can be derived by means of the geneticcode. Alternatively, it is also possible to search for homologoussequences in EST (expressed sequence tags) data bases and combine them.

The nucleic acids according to the invention are suitable for theexpression of eukaryotic neutral sphingomyelinase in prokaryotic oreukaryotic systems. In addition, they are also suitable for expressionof nSMase in vivo in a gene therapy, or especially, in the form offragments with complementary structures, they are also suitable asantisense nucleotides for reducing the expression of nSMase.

The nucleic acids according to the invention can be prepared by chemicalsynthesis or by amplification in genetically engineered organisms bymethods per se known to those skilled in the art.

The invention also relates to eukaryotic neutral sphingomyelinaseobtainable by the expression of the nucleic acids according to theinvention.

The nSMase according to the invention can be prepared by expression ingenetically engineered organisms. Eukaryotic expression systems areparticularly suitable. Appropriate eukaryotic expression systems are tothose skilled in the art, for example, pRc/CMV (Stratagene).Purification from genetically engineered organisms offers an easy anddirect access to the nSMase according to the invention, especially inthe case of overexpression, and in addition allows for the isolationthereof in larger quantities.

The eukaryotic neutral sphingomyelinase is preferably a mammal,especially human or murine, neutral sphingomyellnase. The amino acidsequences of the human and murine neutral sphingomyelinases arerepresented as SEQ. ID. NOS. 1 and 2.

The molecular weights of human and murine sphingomyelinases are 47.6 and47.5 kDa, respectively. In contrast to bacterial nSMases, the mammalnSMases according to the invention do not contain a signal sequence atthe N terminus. From the hydrophobicity analysis, it can be consideredthat two neighboring hydrophobic membrane domains at the C terminus areseparated by eight amino acids. Therefore, the proteins appear to beintegral membrane proteins whose catalytically active domain is directedtowards the cytosol while only a small proportion of the enzymescontacts the extracellular environment. This is in contrast to bacterialnSMases which are secreted, soluble proteins, but in agreement withprevious studies on the properties of neutral sphingomyelinases ofmammals. According to a Northern blot analysis, the 1.7 kb mRNA ofmurine nSMase is expressed in all tissues. In the kidneys, brain, liver,heart and lungs, the Northern blot shows a strong signal whileexpression in the spleen appears to be low. This measurement was not inagreement with the measured enzymatic activities of the correspondingtissues. This speaks in favor of a post-transcriptional regulation ofnSMase.

The pH optimum of the neutral sphingomyelinase according to theinvention is within a range of from 6.5 to 7.5, with a K_(m) value forC18 sphingomyelin within a range of from 1.0 to 1.5×10⁻⁵ M. The activityis dependent on the presence of magnesium ions; the addition of EDTAresults in an inhibition of SMase activity, which can be restored,however, by the addition of Mn²⁺ or Mg²⁺ ions. The addition of 0.3 to0.5% Triton X-100 increases the enzymatic activity. The activity is notaffected by a treatment with DTT or 2-mercaptoethanol whereas theaddition of 20 mM glutathione led to inhibition. The activity of nSMaseis not restricted to sphingomyelin; the structurally relatedphosphatidylcholine was also cleaved with about 3% activity.

Also claimed are variants of the eukaryotic neutral sphingomyellnase.The term “variants” encompasses both naturally occurring allelicvariations of the eukaryotic neutral sphingomyelinase and proteinsprepared by recombinant DNA technology (especially by in-vitromutagenesis using chemically synthesized oligonucleotides) followed byexpression which correspond to eukaryotic neutral sphingomyellnase interms of biological and/or immunological activity. This may include thedeletion, insertion or conservative substitution of amino acids.“Conservative substitutions” means that an amino acid is substituted byanother amino acid having similar physicochemical properties.

Thus, for example, the following amino acids are interchangeable: serineand alanine; alanine and glycine; methionine and serine; lysine andarginine; lysine and serine.

In particular, the term “variants” also includes N-terminally and/orC-terminally truncated proteins as well as acetylated, glycosylated,amidated and/or phosphorylated derivatives.

At least part of the activity of nSMase seems to reside in theC-terminal region since the fragment 1-282 of murine nSMase failed toexhibit an increase of sphingomyelinase activity when expressed inHEK293 cells. This invention also relates to C-terminal fragments ofnSMase. Compounds in which nSMase or its variants are coupled with othermolecules, such as dyes, radionuclides or affinity components, are alsovariants according to the invention.

Also claimed are nucleic acids coding for eukaryotic neutralsphingomyelinase or being complementary to such nucleic acids. Thenucleic acids may be, for example, DNA, RNA, PNA or nuclease-resistantanalogues thereof. In particular, nuclease-resistant analogues includethose compounds which have the phosphodiester linkage modified byhydrolysis-stable compounds, such as phosphothioates, methylphosphonatesor the like.

Especially short fragments of the nucleic acids are suitable asantisense nucleotides. For reasons of specificity, they shouldpreferably contain more than 6, more preferably more than 8 and mostpreferably more than 12 nucleotides. For reasons of diffusion and costs,they usually have a length of less than 30 nucleotides, preferably 24 orless, and more preferably 18 or less nucleotides.

The invention also relates to derivatives of nucleic acids which arecoupled to other molecules for diagnostic or therapeutic purposes, forexample, to fluorescent dyes, radioactive labels or affinity components,and fragments of the nucleic acids according to the invention, and thenucleic acids complementary to these nucleic acids, and variants of thenucleic acids. “Fragments” as used herein means nucleic acids truncatedat the 5′ or 3′ or at both ends. The term “variants” means that thesenucleic acids will hybridize with the nucleic acid according to theinvention or with nucleic acids complementary thereto under stringentconditions. The term “stringent conditions” means that the hybridizationis performed under conditions in which the temperature is even lower byup to 10° C. than the temperature (conditions being otherwise identical)just low enough for exactly complementary nucleic acids to anneal. Forexample, if an exactly complementary nucleic acid will anneal down to atemperature of about 55° C. under given conditions, then stringentconditions are temperatures of equal to or higher than 45° C.Preferably, the temperature range for stringent conditions is within 5°C., more preferably within 3° C.

Further, the invention relates to antibodies directed against the nSMaseaccording to the invention or the nucleic acids according to theinvention.

These substances are suitable, in particular, for use in diagnostics, inimmuno-assays per se known to those skilled in the art, for histologicalstudies and as medicaments for the treatment of conditions associatedwith an overexpression of nSMase. Such antibodies according to theinvention can be obtained by methods per se known to those skilled inthe art through immunization with nSMase, nucleic acids according to theinvention or peptide and nucleic acid fragments in the presence ofadjuvants.

Further, the invention relates to cell lines which overexpress thenSMase according to the invention. Such cell lines can be obtained bytransfection with vectors containing the nucleic acids according to theinvention coding for nSMase. In the case of eukaryotic cell lines, forexample, transfection may be effected by electroporation. Preferably,the cell lines are stably transfected.

In this connection, “overexpression” means that the cell line has ahigher activity of nSMase than cell lines which have not beentransfected with the nucleic acids according to the invention. Forexample, suitable eukaryotic cell lines include the cell lines U937, HEK293 or Jurkat.

In experiments, the cell lines exhibited a specific nSMase activity ofbetween 0.3 and 10 μmol/mg of protein/hour.

FIG. 3 shows the Northern and Western blot analysis of nSMase expressionin transfected cell lines. Portion A shows the result of a RT PCR of thewhole cell RNA with primers hybridizing with human and murine nSMasecDNAs. Portion B shows the T PCR of the whole RNA with primershybridizing with human β-actin cDNA as a control. Portion C shows theWestern blot of the plasma membrane protein extract of different HEK 293cell lines after SDS polyacrylamide gel electrophoresis andhybridization with polyclonal anti-nSMase antibodies.

The addition of 0.5 mM arachidonic acid resulted in a threefold increaseof nSMase activity in the overexpressing HEK cells.

The invention further relates to a transgenic mammal which exhibits anoverexpression (gain of function) or a genetic deficiency or defect(loss of function) for the nSMase according to the invention. The mammalis preferably a rodent, especially a mouse. Such transgenic mammals canbe obtained by methods per se known to those skilled in the art and areespecially suitable for elucidating the function of neutralsphingomyelinase. For transgenic mammals, defined gene constructs areinjected into the pronucleus of a fertilized egg cell by DNAmicroinjection to achieve the expression of an additional gene. Byselectively changing a gene in the genome of ES cells which aresubsequently injected in blastocysts, the function of a gene is switchedoff.

The strategy and constructs for generating the mouse mutants are shownin FIGS. 4 and 5.

The transgenic animals are preferably animals in which the gene can beswitched on and off temporally and in a tissue-specific way by externalinduction. Such transgenic mammals are especially suitable forelucidating the metabolic and signal transduction pathways related tothe nSMase according to the invention; this in turn enables diagnosticor therapeutic applications. In particular, the transgenic mammals aresuitable for the screening of pharmaceutically active substances.

The eukaryotic neutral sphingomyelinase according to the invention, thenucleic acids according to the invention and the antibodies according tothe invention can be contained in medicaments and diagnostic agents,optionally together with further auxiliary agents. Such medicaments anddiagnostic agents are suitable for the diagnosis and treatment ofdiseases based on over- or underexpression and/or an increased orreduced activity of eukaryotic neutral sphingomyelinase and/or disordersof cell proliferation, cell differentiation and/or apoptosis.

In particular, these are diseases in which inflammation processes, cellgrowth disorders and metabolic disorders are involved. For example, theymay be cancers or disorders of cholesterol homeostasis(atherosclerosis).

A pharmaceutical screening method according to the invention relies on achange of the expression or activity of the nSMase according to theinvention in nSMase-overexpressing cell lines upon the addition of atleast one potential pharmaceutically active substance. Thus, the celllines are suitable, in particular, for developing and testingpharmaceutical leading structures.

The invention will be further illustrated by the following Examples.

EXAMPLE 1 Cloning of the Nucleic Acid

The inventive nucleic acids coding for neutral sphingomyelinase werecloned into the NotI restriction sites of the cloning site of theeukaryotic expression vector pRc/CMV (Stratagene). The sequences of theresulting DNAs were obtained by sequencing using a Perkin-Elmer DNAsequencer 377A.

EXAMPLE 2 Cloning of the RNA

The whole RNA was isolated from different organs of eight three-week-oldCD1 mice according to known methods, and poly(A⁺) RNA was isolated byaffinity purification on oligo(dT) cellulose (Boehringer Mannheim,Germany) according to standard methods.

EXAMPLE 3 Overexpressing Cell Lines

U937 cells were grown in PRMI 1640 medium with 10% fetal calf serum, 1μg/ml penicillin/streptomycin and 0.03% glutamine at 37° C. and 5% CO₂.By electroporation with a Gene Pulser (Bio-Rad), 5×10⁶ cells weretransfected with 1 μg of linearized plasmid DNA coding for the nSMaseaccording to the invention. The selection of stable clones was effectedby using 1 mg/ml geneticin (G418, Life Technologies, Gaithersburg, Md.).

The nSMase purified from the cell lines exhibited a specific activity ofbetween 0.3 and 10 μmol/mg of protein/hour. Its pH optimum was at 6.5and 7.5. The K_(M) value for C18 sphingomyelin was from 1.0 to 1.5×10⁻⁵M. The activity was dependent on the presence of magnesium ions; theaddition of EDTA inhibited the activity.

EXAMPLE 4 Measurement of nSMase Activity

The enzymatic activity was examined in cells and murine tissues. Thecells were washed twice with ice-cold PBS and sedimented at 1,000×g. Thepellet was resuspended in lysis buffer, and the cells were disrupted byrepeated cycles of freezing and thawing. After centrifugation at 2,500×gfor 2 min, extraction with lysis buffer containing 0.2% Triton X-100 wasperformed, followed by centrifugation at 100,000×g for 15 min.

Tissue from three-week-old mice was homogenized in cold lysis buffer.The quantity of protein or homogenized tissue to be examined wasincubated with 10 nM (80,000 dpm) [N-¹⁴CH₃]sphingomyelin for 30 min at37° C. in a total volume of 200 μl. Then, 100 μl of water was added, andunreacted substrate was removed by extraction with chloroform-methanol(2:1, v/v). The radioactivity of the aqueous phase containing theenzymatically released phosphocholine was measured in a scintillationcounter.

EXAMPLE 5 Polyclonal Antibodies

Rabbits were immunized with the synthetic peptide CDPHSDKPFSDHE(corresponding to amino acids 261 through 273 of murine nSMase), coupledto keyhole limpet hemocyanin. The polyclonal antibody serum was purifiedby chromatography on hydroxyapatite and affinity chromatography on acolumn having the above mentioned synthetic peptide bound thereto.

6 1 423 PRT Homo sapiens 1 Met Lys Leu Asn Phe Ser Leu Arg Leu Arg IlePhe Asn Leu Asn Cys 1 5 10 15 Trp Gly Ile Pro Tyr Leu Ser Lys His ArgAla Asp Arg Met Arg Arg 20 25 30 Leu Gly Asp Phe Leu Asn Gln Glu Ser PheAsp Leu Ala Leu Leu Glu 35 40 45 Glu Val Trp Ser Glu Gln Asp Phe Gln TyrLeu Arg Gln Lys Leu Ser 50 55 60 Pro Thr Tyr Pro Ala Ala His His Phe ArgSer Gly Ile Ile Gly Ser 65 70 75 80 Gly Leu Cys Val Phe Ser Lys His ProIle Gln Glu Leu Thr Gln His 85 90 95 Ile Tyr Thr Leu Asn Gly Tyr Pro TyrMet Ile His His Gly Asp Trp 100 105 110 Phe Ser Gly Lys Ala Val Gly LeuLeu Val Leu His Leu Ser Gly Met 115 120 125 Val Leu Asn Ala Tyr Val ThrHis Leu His Ala Glu Tyr Asn Arg Gln 130 135 140 Lys Asp Ile Tyr Leu AlaHis Arg Val Ala Gln Ala Trp Glu Leu Ala 145 150 155 160 Gln Phe Ile HisHis Thr Ser Lys Lys Ala Asp Val Val Leu Leu Cys 165 170 175 Gly Asp LeuAsn Met His Pro Glu Asp Leu Gly Cys Cys Leu Leu Lys 180 185 190 Glu TrpThr Gly Leu His Asp Ala Tyr Leu Glu Thr Arg Asp Phe Lys 195 200 205 GlySer Glu Glu Gly Asn Thr Met Val Pro Lys Asn Cys Tyr Val Ser 210 215 220Gln Gln Glu Leu Lys Pro Phe Pro Phe Gly Val Arg Ile Asp Tyr Val 225 230235 240 Leu Tyr Lys Ala Val Ser Gly Phe Tyr Ile Ser Cys Lys Ser Phe Glu245 250 255 Thr Thr Thr Gly Phe Asp Pro His Ser Gly Thr Pro Leu Ser AspHis 260 265 270 Glu Ala Leu Met Ala Thr Leu Phe Val Arg His Ser Pro ProGln Gln 275 280 285 Asn Pro Ser Ser Thr His Gly Pro Ala Glu Arg Ser ProLeu Met Cys 290 295 300 Val Leu Lys Glu Ala Trp Thr Glu Leu Gly Leu GlyMet Ala Gln Ala 305 310 315 320 Arg Trp Trp Ala Thr Phe Ala Ser Tyr ValIle Gly Leu Gly Leu Leu 325 330 335 Leu Leu Ala Leu Leu Cys Val Leu AlaAla Gly Gly Gly Ala Gly Glu 340 345 350 Ala Ala Ile Leu Leu Trp Thr ProSer Val Gly Leu Val Leu Trp Ala 355 360 365 Gly Ala Phe Tyr Leu Phe HisVal Gln Glu Val Asn Gly Leu Tyr Arg 370 375 380 Ala Gln Ala Glu Leu GlnHis Val Leu Gly Arg Ala Arg Glu Ala Gln 385 390 395 400 Asp Leu Gly ProGlu Pro Gln Pro Ala Leu Leu Leu Gly Gln Gln Glu 405 410 415 Gly Asp ArgThr Lys Glu Gln 420 2 419 PRT Murine sp. 2 Met Lys Leu Asn Phe Ser LeuArg Leu Arg Val Phe Asn Leu Asn Cys 1 5 10 15 Trp Asp Ile Pro Tyr LeuSer Lys His Arg Ala Asp Arg Met Lys Arg 20 25 30 Leu Gly Asp Phe Leu AsnLeu Glu Asn Phe Asp Leu Ala Leu Leu Glu 35 40 45 Glu Val Trp Ser Glu GlnAsp Phe Gln Tyr Leu Arg Gln Arg Leu Ser 50 55 60 Leu Thr Tyr Pro Asp AlaHis Tyr Phe Arg Ser Gly Met Ile Gly Ser 65 70 75 80 Gly Leu Cys Val PheSer Lys His Pro Ile Gln Glu Ile Phe Gln His 85 90 95 Val Tyr Ser Leu AsnGly Tyr Pro Tyr Met Phe His His Gly Asp Trp 100 105 110 Phe Cys Gly LysSer Val Gly Leu Leu Val Leu Arg Leu Ser Gly Leu 115 120 125 Val Leu AsnAla Tyr Val Thr His Leu His Ala Glu Tyr Ser Arg Gln 130 135 140 Lys AspIle Tyr Phe Ala His Arg Val Ala Gln Ala Trp Glu Leu Ala 145 150 155 160Gln Phe Ile His His Thr Ser Lys Asn Ala Asp Val Val Leu Leu Cys 165 170175 Gly Asp Leu Asn Met His Pro Lys Asp Leu Gly Cys Cys Leu Leu Lys 180185 190 Glu Trp Thr Gly Leu His Asp Ala Phe Val Glu Thr Glu Asp Phe Lys195 200 205 Gly Ser Asp Asp Gly Cys Thr Met Val Pro Lys Asn Cys Tyr ValSer 210 215 220 Gln Gln Asp Leu Gly Pro Phe Pro Ser Gly Ile Arg Ile AspTyr Val 225 230 235 240 Leu Tyr Lys Ala Val Ser Glu Phe His Val Cys CysGlu Thr Leu Lys 245 250 255 Thr Thr Thr Gly Cys Asp Pro His Ser Asp LysPro Phe Ser Asp His 260 265 270 Glu Ala Leu Met Ala Thr Leu Tyr Val LysHis Ser Pro Pro Gln Glu 275 280 285 Asp Pro Cys Thr Ala Cys Gly Pro LeuGlu Arg Ser Asp Leu Ile Ser 290 295 300 Val Leu Arg Glu Ala Arg Thr GluLeu Gly Leu Gly Ile Ala Lys Ala 305 310 315 320 Arg Trp Trp Ala Ala PheSer Gly Tyr Val Ile Val Trp Gly Leu Ser 325 330 335 Leu Leu Val Leu LeuCys Val Leu Ala Ala Gly Glu Glu Ala Arg Glu 340 345 350 Val Ala Ile IleLeu Cys Ile Pro Ser Val Gly Leu Val Leu Val Ala 355 360 365 Gly Ala ValTyr Leu Phe His Lys Gln Glu Ala Lys Gly Leu Cys Arg 370 375 380 Ala GlnAla Glu Met Leu His Val Leu Thr Arg Glu Thr Glu Thr Gln 385 390 395 400Asp Arg Gly Ser Glu Pro His Leu Ala Tyr Cys Leu Gln Gln Glu Gly 405 410415 Asp Arg Ala 3 1662 DNA Homo sapiens 3 gcggccgcga ccgccggggacgagcttgga ggaaaaggaa ccgggagccg cccacccggg 60 ggcgctctcc ggacccccagggtcctagcg cgcggccctt accgagcctg ggcgcccgga 120 tttcggsagc ggatcgcctttccgggttgg cggcccgcct gattgggaac agccggccgg 180 ttgccggggg aacgcgggagtcgggcccga cctgagccac gcgggcttgg tgcccacctg 240 tgcgcgccgc ctgcgaagaaggaacggtct agggagaagg cgccgccggc cgcccccgtc 300 cccaccgcgg ccgtcgctggagagttcgag ccgcctagcg cccctggagc tccccaacca 360 tgaagctcaa cttctccctgcgactgcgga tcttcaacct caactgctgg ggcattccgt 420 acttgagcaa gcaccgggccgaccgcatga ggcgcctggg agactttctg aaccaggaga 480 gcttcgacct ggctttgctggaggaggtgt ggagtgagca ggacttccag tacctgagac 540 agaagctgtc acctacctacccagctgcac accacttccg gagcggaatc attggcagtg 600 gcctctgtgt cttctccaaacatccaatcc aggagcttac ccagcacatc tacactctca 660 atggctaccc ctacatgatccatcatggtg actggttcag tgggaaggct gtggggctgc 720 tggtgctcca tctaagtggcatggtgctca acgcctatgt gacccatctc catgccgaat 780 acaatcgaca gaaggacatctacctagcac atcgtgtggc ccaagcttgg gaattggccc 840 agttcatcca ccacacatccaagaaggcag acgtggttct gttgtgtgga gacctcaaca 900 tgcacccaga agacctgggctgctgcctgc tgaaggagtg gacagggctt catgatgcct 960 atcttgaaac tcgggacttcaagggctctg aggaaggcaa cacaatggta cccaagaact 1020 gctacgtcag ccagcaggagctgaagccat ttccctttgg tgtccgcatt gactacgtgc 1080 tttacaaggc agtttctgggttttacatct cctgtaagag ttttgaaacc actacaggct 1140 ttgaccctca cagtggcacccccctctctg atcatgaagc cctgatggct actctgtttg 1200 tgaggcacag ccccccacagcagaacccca gctctaccca cggaccagca gagaggtcgc 1260 cgttgatgtg tgtgctaaaggaggcctgga cggagctggg tctgggcatg gctcaggctc 1320 gctggtgggc caccttcgctagctatgtga ttggcctggg gctgcttctc ctggcactgc 1380 tgtgtgtcct ggcggctggaggaggggccg gggaagctgc catactgctc tggaccccca 1440 gtgtagggct ggtgctgtgggcaggtgcat tctacctctt ccacgtacag gaggtcaatg 1500 gcttatatag ggcccaggctgagctccagc atgtgctagg aagggcaagg gaggcccagg 1560 atctgggccc agagcctcagccagccctac tcctggggca gcaggagggg gacagaacta 1620 aagaacaata aagcttggccctttaaaaaa aaaaaaaaaa aa 1662 4 1627 DNA Murine sp. 4 gtgctggtggaagccgagcc gggaacaagg gaggaacctg taggccgcgg tgcgagaacc 60 caccgaagacctaagaatct ggaacagtcc acccgagatt ccttccagga ctgccggcgg 120 ctcgcgcaccagcccgggat ttgcagccga ccttctttcc gggtggaagg acggcctttg 180 tcccagtaacgcaggagtcg ccccccaccc ccaaccagct cgcgttcctg ggtcggggca 240 gcgcaggacagggcaataag cctgtgcgcg caatccgcct cgccgccctt gctccgaagc 300 actccagccatgaagctcaa cttttctcta cggctgagag ttttcaatct caactgctgg 360 gacatcccctacctgagcaa acatagggcg gaccgcatga agcgcttggg agactttctg 420 aacttggaaaactttgatct ggctctcctg gaggaggtgt ggagtgagca ggacttccag 480 tacctaaggcaaaggctatc gctcacctat ccagatgcac actacttcag aagcgggatg 540 ataggcagtggcctctgtgt gttctccaaa cacccaatcc aggaaatctt ccagcatgtc 600 tacagtctgaatggttaccc ctacatgttc catcatggag actggttctg tgggaagtct 660 gtggggctgctggtgctccg tctaagtgga ctggtgctca atgcctacgt gactcatcta 720 catgctgagtacagccgaca gaaggacatc tactttgcac accgtgtggc ccaagcttgg 780 gaactggcccagttcatcca ccacacatcc aagaatgcag atgtggttct attgtgtgga 840 gacctcaatatgcaccccaa agacctgggc tgctgcctgc tgaaagagtg gacagggctc 900 catgatgctttcgttgagac tgaggacttt aagggctctg atgatggctg taccatggta 960 cccaagaactgctacgtcag ccagcaggac ctgggaccgt ttccgtctgg tatccggatt 1020 gattacgtgctttacaaggc agtctctgag ttccacgtct gctgtgagac tctgaaaacc 1080 actacaggctgtgaccctca cagtgacaag cccttctctg atcacgaggc cctcatggct 1140 actttgtatgtgaagcacag cccccctcag gaagacccct gtactgcctg tggcccactg 1200 gaaaggtccgatttgatcag cgtgctaagg gaggccagga cagagctggg gctaggcata 1260 gctaaagctcgctggtgggc tgcattctct ggctatgtga tcgtttgggg gctgtccctt 1320 ctggtgttgctgtgtgtcct ggctgcagga gaagaggcca gggaagtggc catcatcctc 1380 tgcatacccagtgtgggtct ggtgctggta gcaggtgcag tctacctctt ccacaagcag 1440 gaggccaagggcttatgtcg ggcccaggct gagatgctgc acgttctgac aagggaaacg 1500 gagacccaggaccgaggctc agagcctcac ctagcctact gcttgcagca ggagggggac 1560 agagcttaagagcttaacaa taaaacttgc ttgacacaca aaaaaaaaaa aaaaaaaaaa 1620 aaaaaaa 16275 4464 DNA Homo sapiens modified_base (2435) a, t, c, g, other orunknown 5 gactcgatcc ccgcgaacgc tcgctcgcgc tccgagtctc ttccaggtcgcccttccttg 60 cgaccagcat ttgttctcta tgcccccatc cagccctagg acagaacgtggacccccgcc 120 cgccagcgca ggcgacaccg cggcaggggg ctgaggtgcg cacggcgtctggggcgaggg 180 gttacctcag cgatggtctt tgacacctga aagctggagc ttttgaagagccccaccacc 240 ttcagcttca ggggcggctc gggcggcaac cgcacgtgac atgctgggggcttcgacttg 300 ggccggcacg gctgctgggt ggccatggca gggacagcag agagcccggaacacaaatag 360 tgcgagtcgc cagggcaacc gcgtggctcc tccccgaacg cccgcaaggggcgggacctg 420 agtgagttcg tgggcggggc ctcgcatcaa cttcaagcct gttgctggtggaagccgagc 480 cgggaacaag ggaggaacct gtaggccgcg gtgcggataa cccaccgaaggacctaagaa 540 tctggaacag tccacccgag attccttcca ggactgccgg cggactctcgcattcagccc 600 gggatttgca gccgaccttc tttccgggtg gaatgacggc ctttgtcccagtaacgcagg 660 agtagccccc cacccccaac cagctcgcgt tcctgggtcg gggcagcgcaggatagggca 720 ataagcctgt gcgcgcaatc cgcctcgccg cccttgctcc gaagcactccagccatgaag 780 ctcaactttt ctctacggct gagagttttc aatctcaact gctggtaagtaagtgctccc 840 aggcgtgggc tgcagcctcg gagccacctt ccagtcccct ctcgcacatgcctaggaagg 900 aagcaggtct tcttcagccg agctagaccc tgtccttccc gaaccaccaaagtccacatc 960 gcctaaagac cagagcttgg gtggttgcag caatcaccaa agtccctatcatccaaagct 1020 gaggtgatga cagcagtaat cgtcccaaac ctggcccatg tctttccttttaaatgattt 1080 acttttattt tatgtacatt tggtgttttg cctgtatgta tgtctgtgtgaaggtgccag 1140 attctctgga actggagtta cagacagttg taagctgtca tgtgcttgctggaaattgaa 1200 ctgctgaccc atctcttctg ccccctgcgt cctccacccc ttttagggacatcccctacc 1260 tgagcaaaca tagggcggac cgcatgaagc gcttgggaga ctttctgaacttggaaaact 1320 ttgatctggc tctcctggag gaggtgaggt tgtagggcag gctaggttggaggagggcag 1380 caggcggcag gcggcggcag gaaaacttgt tctgtcttgg gatgaaatcccaagcaagta 1440 tcctcacctt cttcctccag gtgtggagtg agcaggactt ccagtacctaaggcaaaggc 1500 tatcgctcac ctatccagat gcacactact tcagaaggtg aaaagcctgtgttctcagcc 1560 tgttctcaga cgaggaagct ctccaacatt cttgcttgca ccctcgatcttcttcctctg 1620 ggtgtgagaa gagcaggccg tcaccctcat cttgcaaggg ctgctgtcttaggctttgtt 1680 ctggggttga tcttagcagt agagctggga gaccgcggag gggaagagggctggctgggt 1740 actcccctcc ttgctcttct ggttattaag caagagttgg ttttcagcgggatgataggc 1800 agtggcctct gtgtgttctc caaacaccca atccaggaaa tcttccagcatgtctacagt 1860 ctgaatggtt acccctacat ggtaaggatc tcttccctat ccttgctaacacagactgga 1920 cgcagccttc ctggggcctt ggcaggaggg tgtcagtacc ctgagtttttgtcttctctt 1980 gcctgcagtt ccatcatgga gactggttct gtgggaagtc tgtggggctgctggtgctcc 2040 gtctaagtgg actggtgctc aatgcctacg tgactcatgt gagtggggctagccaggctt 2100 aggcagtggg tcaagcagcc caatgctatg gtggagaaga gacgccactagttagttctg 2160 ctgcctgggg ataaggcatg ggatcagaag ctagcattgg gcaaggttcacccattccct 2220 gtcacactct gccatgtgac agatgacaag cttgattcag acagccttctctttgatttc 2280 acctattcca ctttagctac atgctgagta cagccgacag aaggacatctactttgcaca 2340 ccgtgtggcc caagcttggg aactggccca gttcatccag tgtgtgagcctgggcttgat 2400 gggggctgtg gggtggggac ggggttgagg gatgngnaan ttatccttgaagagggcaca 2460 taataaggga agaatttcct ccttgccgct cttcccccaa ctcagccacacatccaagaa 2520 tgcagatgtg gttctattgt gtggagacct caatatgcac cccaaagacctgggctgctg 2580 cctgctgaaa gagtggacag ggctccatga tgctttcgtt gagactgaggactttaaggt 2640 gagagactgt ttcccaccaa ctccacactt gttccagtct tcctgtctcttagcatccta 2700 gccacctgtt tccctagggc tctgatgatg gctgtaccat ggtacccaagaactgctacg 2760 tcagccagca ggacctggga ccgtttccgt ctggtatccg gattgattacgtgctttaca 2820 aggtcaggct cttattcccg gtgtgccttc tccagtatct tccttcctctgtcactagcc 2880 cacgctttag ttcagctaca gtcttgggcc actgatggct aaagaatagaatcctgtcgg 2940 ctggttctct gggagaattt aagcttctcc atgttcttgc tcttcctaggcagtctctga 3000 gttccacgtc tgctgtgaga ctctgaaaac cactacaggc tgtgaccctcacagtgacaa 3060 gcccttctct gatcacgagg ccctcatggc tactttgtat gtgaagcacagcccccctca 3120 ggaagacccc tgtactgcct gtggtaagca gcatttcctt tgccccctctactttaaggc 3180 agccccgcct ccatcctgac cctcccctgc tctacgttct ctctttttccaggcccactg 3240 gaaaggtccg atttgatcag cgtgctaagg gaggccagga cagagctggggctaggcata 3300 gctaaagctc gctggtgggc tgcattctct ggctatgtga tcgtttgggggctgtccctt 3360 ctggtgttgc tgtgtgtcct ggctgcagga gaagaggcca gggaagtggccatcatcctc 3420 tgcataccca gtgtgggtct ggtgctggta gcaggtgcag tctacctcttccacaagcag 3480 gaggccaagg gcttatgtcg ggcccaggct gagatgctgc acgttctgacaagggaaacg 3540 gagacccagg accgaggctc agagcctcac ctagcctact gcttgcagcaggagggggac 3600 agagcttaag agcttaacaa taaaacttgc ttgacacact ctagtggctctaccttgttc 3660 cttgcagagg catgatggga actgaaggtc agtggccttg tcactgtgtggctttagagc 3720 gttggcctct cacttgcctt ttttgcacac tcccgtctcc tgccagcacagagcataaac 3780 cctgttcatg gtcataatcc ttttattgta aacaacgaag cctctgactaagcagtccag 3840 atggcggagg tacagccctt gtgatggtgt cttgcttacg gggcagggaggcagctaacc 3900 atcatcttct agccctgggc tcccatctat gcaggcatct ctctgagcctccgttcctcc 3960 tggaattggn tcagagcaat cccgcttggt tcaccaacct ccaaacagcttccttaagga 4020 cctggtttct caaaanggna aggtncgggc ctccggtctt caatangttttcctaaaaag 4080 ggangaatga aaanccttaa gnnccaacaa ggggaaccct tggncccaaaaggggacctg 4140 ggtggtttcc cnttggggcc aaanttatcc caaaggggtc caattgaagggttaaccccc 4200 caaaaannac ccntttcccc cggaatttcc aaaggtttnc cccccccggcaaaanctccc 4260 ttggggnncc naanccntgg cccggncttg gcttttcccc ctttcccaagnatttcaaan 4320 nttccctngg aaancccctt gnttggnaaa accnaatnan gaaccangccaannnttgcc 4380 aanaaaccnt ttgggcaaag ggggnaaatt cancaanggg gnaattggggaaacccntgg 4440 gtttncccaa agggcccnaa nant 4464 6 2852 DNA Murine sp.modified_base (1949) a, t, c, g, other or unknown 6 accgcggccgtcgctggaga gttcgagccg cctagcgccc ctggagctcc ccaaccatga 60 agcccaacttctccctgcga ctgcggatct tcaacctcaa ctgctggtga gtgcgtctgc 120 ggagtgcggtctgggggcca ccttccgttc gcacccatgc agccttcctc cccctatccc 180 gccccacgatctcagggtgt agggaaaacc cgaacctcca aagtccacat ctggccccag 240 cgccggtggtcccagcagtc gcctcccctg ccccgctctt cccttcctta ggggcattcc 300 gtacttgagcaagcaccggg ccgaccgcat gaggcgcctg ggagactttc tgaaccagga 360 gagcttcgacctggctttgc tggaggaggt gagattgtgc agcacggtgc ggaacccagg 420 ctgggaggagggacagaccg tcccactggg gaaagaccaa gcaggcatcc tcaccgcttc 480 cctcaggtgtggagtgagca ggacttccag tacctgagac agaagctgtc acctacctac 540 ccagctgcacaccacttccg gaggtgagaa gcccactggc ctgaagcctg ttgtcatccc 600 aggaggctcttggccctgcc agcccttccc tatcctgcct gcactctcca gtctcctcca 660 gcctcctctccctctggatg tgagagaagg agaagggtga accaagaagg tcctatgact 720 tcagcccatttcagctttgt tttctggctg ccctatactc ctccaaaggc cgtcgccttg 780 gttctagggctagtcccagc agtagaaaaa gaaaaaaata gctgatcaga gctggaagac 840 aagggaggggaagaaggctg ggtgtctctc cctgtttttc tggttattaa gcagggcttg 900 gctttcagcggaatcattgg cagtggcctc tgtgtcttct ccaaacatcc aatccaggag 960 cttacccagcacatctacac tctcaatggc tacccctaca tggtaaggca gacctttgac 1020 ctcttccacctcccttcccc acctccagta atacaaggta gaggaggcag ccctctgaga 1080 gctgcaggggatgggcagaa agatggtggc ggtgccctga gtttctatct cctcctgcct 1140 gcagatccatcatggtgact ggttcagtgg gaaggctgtg gggctgctgg tgctccatct 1200 aagtggcatggtgctcaacg cctatgtgac ccatgtgagt gaagctggca gtgcctaggg 1260 ctgggacatgcagcccagtc ctgggacaga gagatggtac ttctctagct ctcatacctg 1320 gggatgaggtgtgggggcaa gatcttataa ggaagcaatg ggcaaggctt atccattgta 1380 taccaaacaccatgccaagt gacagacaca ggcttgattc agacataccc ctgggaccct 1440 cagtcttatctgctgtgatc tcatccatct tgctcagctc catgccgaat acaatcgaca 1500 gaaggacatctacctagcac atcgtgtggc ccaagcttgg gaattggccc agttcatcca 1560 gtgtgtgagcctgggcttga aatgggaagt gggatgggac ccaggggctg agggtgaaca 1620 aggccccagtcatggggaag agctggtgat ggaagaactc ccgcctcacc aacctggttc 1680 ccccagccacacatccaaga aggcagacgt ggttctgttg tgtggagacc tcaacatgca 1740 cccagaagactgggctgctg cctgctgaag gagtggacag ggcttcatga tgcctatctt 1800 gaaactcgggacttcaaggt gaggacttgc ctgttacttc cccacctata tccccagctt 1860 ctctccctccttctccccca catcctagca tgagccaatg attcccttag ggctctgagg 1920 aaggcaacacaatggtaccc aagaactgnt acgtcagcca gcaggagctg aagccatttc 1980 cctttggtgtccgcattgac tacgtgcttt acaaggtcag gctcctccct tcaacatgct 2040 ttcatatgctgtgtctcttt gtctactaac ctgtgtagat cctttgctca gntagtctag 2100 tcttggaccactgatgggtg gaaagtgggg tagccgggag ctggttctct gggaagaggc 2160 cctcatatataagcttctct ntggccctta cttttcctag gcagtttctg ggttttacat 2220 ctcctgtaagagttttgaaa ccactacagg ctttgaccct nacaggggca cccccctctc 2280 ttgatcatgaagccctgatg gctactctgt ttgtgaggca cagcccccca cagcagaacc 2340 ccagctctacccacggtgag tcacccccac cctttccttg gcccttgccc cgcttgaagc 2400 agcccttccactcttgactc tctcctgccc cactgccctg ctctgttgta ggaccagcag 2460 agaggtcgccgttgatgtgt gtgctaaagg aggcctggac ggagctgggt ctgggcatgg 2520 ctcaggctcgctggtgggcc accttcgcta gctatgtgat tggcctgggg ctgcttctcc 2580 tggcactgctgtgtgtcctg gcggctggag gaggggccgg ggaagctgcc atactgctct 2640 ggacccccagtgtagggctg gtgctgtggg caggtgcatt ctacctcttc cacgtacagg 2700 aggtcaatggcttatatagg gcccaggctg agctccagca tgtgctagga agggcaaggg 2760 aggcccaggatctgggccca gagcctcagc cagccctact cctggggcag caggaggggg 2820 acagaactaaagaacaataa agcttggccc aa 2852

What is claimed is:
 1. An isolated eukaryotic neutral sphingomyelinasecomprising a sequence according to a) SEQ ID NO: 1 or SEQ ID NO: 2, orb) a variant of SEQ ID NO: 1 or SEQ ID NO: 2 having eukaryotic neutralsphingomyelinase enzymatic activity, wherein the variant is i) anaturally occurring allelic variation of SEQ ID NO: 1 or SEQ ID NO: 2,or ii) SEQ ID NO: 1 or SEQ ID NO: 2 having one inserted, deleted, orconservatively substituted amino acid, or iii) An N-terminally truncatedor C-terminally truncated SEQ ID NO: 1 or SEQ ID NO: 2, or iv) anacetylated, glycosylated, amidated, or phosphorylated naturallyoccurring allelic variation of SEQ ID NO: 1 or SEQ ID NO: 2, or v) anacetylated, glycosylated, amidated, or phosphorylated SEQ ID NO: 1 orSEQ ID NO: 2 having one inserted, deleted, or conservatively substitutedamino acid, or vi) an acetylated, glycosylated, amidated, orphosphorylated N-terminally truncated or C-terminally truncated SEQ IDNO: 1 or SEQ ID NO: 2, or vii) an acetylated, glycosylated, amidated,orphosphorylated SEQ ID NO: 1 or SEQ ID NO:
 2. 2. An N-terminallytruncated or C-terminally truncated SEQ ID NO: 1 or SEQ ID NO: 2 havingeukaryotic neutral sphingomyelinase enzymatic activity.
 3. A medicamentcontaining the isolated eukaryotic neutral sphingomyelinase according toclaim 1, together with auxiliary agents.
 4. A diagnostic agentcontaining the isolated eukaryotic neutral sphingomyelinase according toclaim 1, together with auxiliary agents.
 5. The isolated eukaryoticneutral sphingomyelinase of claim 1, wherein the sequence is SEQ ID NO:1 or SEQ ID NO:
 2. 6. The isolated eukaryotic neutral sphingomyelinaseof claim 1, wherein the sequence is a variant of SEQ ID NO: 1 or SEQ IDNO: 2 having eukaryotic neutral sphingomyelinase enzymatic activity,wherein the variant is a naturally occurring allelic variation of SEQ IDNO: 1 or SEQ ID NO:
 2. 7. The isolated eukaryotic neutralsphingomyelinase of claim 1, wherein the sequence is a variant of SEQ IDNO: 1 or SEQ ID NO: 2 having eukaryotic neutral sphingomyelinaseenzymatic activity, wherein the variant is SEQ ID NO: 1 or SEQ ID NO: 2having one inserted, deleted, or conservatively substituted amino acid.8. The isolated eukaryotic neutral sphingomyelinase of claim 1, whereinthe sequence is a variant of SEQ ID NO: 1 or SEQ ID NO: 2 havingeukaryotic neutral sphingomyelinase enzymatic activity, wherein thevariant is -terminally truncated or C-terminally truncated SEQ ID NO: 1or SEQ ID NO:
 2. 9. The isolated eukaryotic neutral sphingomyelinase ofclaim 1, wherein the sequence is a variant of SEQ ID NO: 1 or SEQ ID NO:2 having eukaryotic neutral sphingomyelinase enzymatic activity, whereinthe variant is acetylated, glycosylated, amidated, and/or phosphorylatedSEQ ID NO: 1 or SEQ ID NO: 2.